Fluorescent signal transduction in a self-assembled Hg2+ chemosensor tuned by various interactions in micellar aqueous environment

Article abstract of DOI:10.1016/j.jphotochem.2019.112276

A new OFF-ON fluorescent chemosensor for ions Hg²⁺ in water has been designed by associating fluorophore 10-methylacridinium perchlorate (MA⁺ClO₄ ^?) with a selective lipophilic sulfur-containing ligand, such as N,N-bis (2-hydroxyethylthio-1-ethyl)dodecyl amine (DBSO). The communication between the two subunits is only due to their spatial closeness, ensured by sodium dodecyl sulfate (SDS) micellar aggregates. The quenching of MA⁺ fluorescence is ascribed to a photoinduced electron-transfer from the ligand to the excited fluorophore, still the emission is quickly restored by addition of Hg²⁺ (as chloride salt) due to the complex formation between ligand and metal. The sensitivity of the system improves by decreasing the ligand concentration, as well as by changing the stoichiometry of the ligand-Hg²⁺ complex from 1:1 to 2:1. DFT calculations give interesting insights into the structures of the complexes and provide useful information on the sensorial activity experimentally observed. The replacement of MA⁺ClO₄ ^? with 9-cyanoanthracene, a fluorophore differently localized on the micellar aggregate, highlights the effect of Cl^? coordination to the complexed Hg²⁺ on the operating mechanism of the sensor. These results demonstrate as the micellar aggregates provide for a straightforward means to regulate the response of the sensorial system without altering the recognition or reporter unit.

Full text of DOI:10.1016/j.jphotochem.2019.112276

Journal Pre-proof
Fluorescent signal transduction in a self-assembled Hg2+ chemosensor
tuned by various interactions in micellar aqueous environment
Raimondo Germani, Paola Anastasio, Michele Chiodini, Tiziana Del
Giacco, Matteo Tiecco, Leonardo Belpassi
PII:
S1010-6030(19)31410-8
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112276
JPC 112276
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
16 August 2019
5 November 2019
29 November 2019
Please cite this article as: Germani R, Anastasio P, Chiodini M, Del Giacco T, Tiecco M,
Belpassi L, Fluorescent signal transduction in a self-assembled Hg2+ chemosensor tuned by
various interactions in micellar aqueous environment, Journal of Photochemistry and amp;
Photobiology, A: Chemistry (2019), doi: https://doi.org/10.1016/j.jphotochem.2019.112276
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Fluorescent signal transduction in a self-assembled Hg2+ chemosensor tuned by
various interactions in micellar aqueous environment.
Raimondo Germania,b, Paola Anastasioa, Michele Chiodinia, Tiziana Del Giaccoa,b,*, Matteo Tieccoa
and Leonardo Belpassic
aDipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 8,
06123 Perugia, Italy
bCentro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN), Università di Perugia, Via
Elce di Sotto 8, 06123 Perugia, Italy
cIstituto di Scienze e Tecnologie Molecolari del CNR (CNR-ISTM), Università di Perugia, Via Elce
di Sotto 8, 06123 Perugia, Italy
*Corresponding author. Fax: +39 755855560.
E-mail addresses: tiziana.delgiacco@unipg.it (T. Del Giacco)
Graphical abstract
1

Highligths:
 Assembly of acridinium perchlorate with an aza-thioether by micelles as a new Hg2+ sensor
is focused.
 The fluorescent transduction signal depends on stoichiometry of the aza-thioether -Hg2+
complex.
 The operating switching mechanism is supposed to be a photoinduced electron transfer
process.
Abstract
A new OFF-ON fluorescent chemosensor for ions Hg2+ in water has been designed by associating

fluorophore 10-methylacridinium perchlorate (MA+ClO4 ) with a selective lipophilic sulfur-
containing ligand, such as N,N-bis (2-hydroxyethylthio-1-ethyl)dodecyl amine (DBSO). The
communication between the two subunits is only due to their spatial closeness, ensured by sodium
dodecyl sulfate (SDS) micellar aggregates. The quenching of MA+ fluorescence is ascribed to a
photoinduced electron-transfer from the ligand to the excited fluorophore, still the emission is
2

quickly restored by addition of Hg2+ (as chloride salt) due to the complex formation between ligand
and metal. The sensitivity of the system improves by decreasing the ligand concentration, as well as
by changing the stoichiometry of the ligand-Hg2+ complex from 1:1 to 2:1. DFT calculations give
interesting insights into the structures of the complexes and provide useful information on the

sensorial activity experimentally observed. The replacement of MA+ClO4 with 9-cyanoanthracene,
a fluorophore differently localized on the micellar aggregate, highlights the effect of Cl
coordination to the complexed Hg2+ on the operating mechanism of the sensor. These results
demonstrate as the micellar aggregates provide for a straightforward means to regulate the response
of the sensorial system without altering the recognition or reporter unit.
Keywords: self-assembling, fluorescent sensor, micellar aggregate, photoinduced electron transfer,
complex stoichiometry
1. Introduction
The development of self-assembled fluorescent chemosensors has been attracting considerable
attention in recent years. New efficient strategies have been developed to overcome the synthetic
difficulty of connection between the two main components of a fluorescent sensor, namely receptor
and fluorophore [13]. One of these implies the molecular organization of the sensor components
into a supramolecular assembly, which provides close proximity between the active components,
producing systems with new features. Actually, the possible insertion of lipophilic species inside
hydrophobic core of surfactant aggregates, or binding of ionic species at the oppositely charged
micellar surface, displays great capacity to establish the communication among the active species
(i.e., fluorophore, receptor and analyte) in pure aqueous medium. The association of the active
components of sensorial systems inside micellar nanostructures has been explored in order to design
3

new classes of fluorescent chemosensors with sensitivity and selectivity that are competitive with
those of existing linked sensors [411].
In a previous work, we have exploited the self-assembling strategy for the realization of an off-
on fluorescent PET (photoinduced electron transfer) sensor for Hg2+ [6], one of the most toxic
heavy metal ions for the environment and health [12,13]. This study was finalized to rationalize if
the nature of the components, in terms of hydrophilic/lipophilic balance, was able to tune the
sensing property of the sensor system in the Hg2+ detection. Following this strategy, considering the
growing demand in highly sensitive and selective detection of trace concentration of Hg2+ in the
environment and physiological media [14-17], we have sought to develop a sensorial system that
provides easy detection of the analyte at a largely sensitivity level. For this purpose, in the present
study we have described the properties of associating an efficient fluorescent reporter (cationic: 10-

methylacridinium perchlorate, MA+ClO4 , and non-ionic: 9-cyanoanthracene, CA, Scheme 1) and a
lipophilic sulfur-containing ligand (N,N-bis (2-hydroxyethylthio-1-ethyl)dodecyl amine, DBSO,
Scheme 1), which selectively binds soft Hg2+ ions. Both fluorophores were selected for the
following reasons: (i) the excellent fluorescence and electrochemical properties permit a high
sensitivity [18,19], and (ii) the fluorescence properties are not altered by Hg2+ [20]. In water, the
relatively lipophilic ligand and fluorophore molecules move into SDS inert aggregates to generate a
supramolecular assembly able to detect metal ion concentration well below the micromolar range.
The results herein described emphasize how the fluorophore/receptor molar ratio affects the
sensitivity of the sensorial system, as well as the stoichiometry of the DBSO-Hg2+ complex.
Moreover, the non-ionic and charged nature of two fluorophores, which determines a different
interaction with the micellar pseudo-phase, and their different photophysical and electrochemical
properties play a fundamental role as regards the sensor performance. The transduction is based on
the enhanced fluorescence consequently to the DBSO-Hg2+ interaction, supposedly owing to the
preclusion of PET process between the excited fluorophores and the receptor. This has allowed the
realization of a more desirable “turn-on” sensor to achieve high selectivity [21]. The results of this
4

study have been integrated by DFT (Density Functional Teory) calculations in order to obtain
information on the geometry of the Hg-receptor complexes. This information was useful to better
support the implication of 1:1 and 2:1 (ligand:Hg2+) complexes in the sensing mechanism
switching.

Scheme 1. Molecular structures of fluorophores MA+ClO4 and CA, receptor DBSO and surfactant
SDS.
2. Experimental
2.1 Instrumentation and materials
Absorption spectra were recorded on an Agilent 8453 Diode Array UV-Vis spectrophotometer.
Fluorescence emission spectra were measured with a BioTek Synergy H1 Hybrid Multi-Mode
1
Microplate Reader spectrometer. H NMR spectra were recorded on a Bruker Avance III HD 400
1
MHz Smart Probe spectrometer (400 MHz for H NMR) and were internally referenced to the
residual proton solvent signal.
The organic solvents were of HPLC grade. Milli Q water (18 M, pH 6.50) was used for all
spectroscopic measurements. The measurements were carried on unbuffered aqueous solutions.
Commercially available SDS (Sigma-Aldrich) was recrystallized twice from an acetone–methanol
mixture before using [22]. All the chloride salts of Hg2+, K+, Mg2+, Ca2+, Mn2+, Ni2+, Cu2+, Zn2+,
Cd2+, and Al3+ions and nitrate and perchlorate salts of Ag+, Pb2+ and Mg2+, respectively, purchased
from commercial suppliers, were of analytical reagent grade and used after drying. All the other
5


reagents were obtained from commercial suppliers and used as received. MA+ClO4 was
synthesized from acridine by quaternization with methyl iodide in acetonitrile and subsequent ion

exchange by sodium perchlorate in methanol [23]. Spectral data of MA+ClO4 are in accordance
with literature data [24]. Receptor DBSO was synthesized in three steps following procedures
already known. Firstly, N,N-bis(2-hydroxyethyl)dodecylammonium chloride was prepared by
alkylation of bis(2-hydroxyethyl)amine with dodecyl bromide and then converted in N,N-bis(2-
chloroethyl)dodecylammonium chloride by reaction with thionyl chloride in anhydrous
dichloromethane [25,26]. In the last step, amine hydrochloride (13 mmol), 2-hydroxy-1-ethanethiol
(29 mmol) and K2CO3 (39 mmol) were placed in a round bottom flask fitted with a reflux condenser
and then mixed in 200 mL of acetonitrile. The mixture was magnetically stirred at 75 °C for 24 h
under nitrogen; after cooling, K2CO3 was filtered off and the solvent was removed under reduced
pressure to yield a yellow oil. The product was purified by column chromatography (silica gel)
using diethyl ether as eluent to give a pale yellow liquid (60 % yield). 1HNMR (CDCl3, 400 MHz):
δ 3.77-3.75 (m, 4H, -CH2OH), 277-268 (m, 12H, -NCH2CH2SCH2-), 2.53-2.50 (m, 2H, N-CH2-
(CH2)10CH3); 1.47 (s broad, 2H, NCH2CH2(CH2)9CH3); 1.27 (s, 18H, NCH2CH2(CH2)9CH3); 0.89
(t, 3H, N-(CH2)11CH3).
2.2 Photophysical measurements
All spectrophotometric and spectrofluorometric analyses of the fluorophore/receptor/metal
systems were performed in 0.010 M SDS aqueous solutions at 25.0 °C. The stock solutions of
fluorophores MA+ClO4 (1.0  10-3 M), CA (1.0  10-3 M) and receptor DBSO (0.1 M) were

prepared in water, acetonitrile and ethanol, respectively. The stock solutions of metal ions and
solutions diluted by these were prepared in aqueous solution. Diluted surfactant solutions of
fluorophore and receptor were prepared about 24 hours before spectroscopic measurements, so that
the components had the time to equilibrate between the aqueous bulk and the micellar aggregates.
Acridinium and cyanoanthracene concentrations were kept 5.3  10-6 and 5.0  10-6 M, respectively,
6

in all solutions; this allowed an absorbance < 0.1 at the excitation wavelength (359 and 365 nm,
respectively), necessary to avoid self-absorption effects. Generally, the UV-Vis absorption and
fluorescence titrations were performed by the gradual addition of the metal ion stock solution into
3.2 ml of fluorophore/receptor surfactant solution in a quartz cuvette with 1 cm optical path. All
solutions were mixed carefully before measurements.
2.3 Computational details
The geometry optimizations were computed with the ADF package (version 2016.104) at the
density functional theory (DFT) level using TZ2P Slater-type basis sets [27], Becke’s exchange
functional in combination with the Lee − Yang − Parr correlation functional [28,29], frozen-core
approximation and ZORA Hamiltonian to account relativistic effects [30-32]. We employed the
numerical integration grid with precision 6.0 (Table S1). All of the studied geometries that have
been optimized are local minima.
3. Results and discussion
3.1. Spectrophotometric titration of DBSO with Hg2+ in SDS micelle solutions.
The interaction of DBSO with Hg2+ was first studied by UV-spectroscopy in the absence of
fluorophore to determine the intrinsic behavior of the receptor. The spectrophotometric titration of
DBSO (1.0  10-4 M) upon mixing with Hg2+ (0  3.34  10-4 M) in 0.010 M SDS solution was
reported in Fig. 1. The SDS concentration was maintained higher than its critical micellar
concentration (8.0  10-3 M in pure water [33], but it could be lower in the presence of electrolyte)
in all the experiments. The addition of increasing amounts of metal ions clearly led to a
batochromically shifted absorption around 240 nm, as well as an increase of absorbance up to ∼1 
10-4 M Hg2+ (concentration equal to that of DBSO), which was assigned to the DBSO-Hg2+
complex formation.
7

3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.9
A250
0.6
0.3
0.0
1.0x10-4 2.0x10-4 3.0x10-4
0.0
[Hg2+], M
210
240
270
300
330
360
Wavelength (nm)
Fig. 1. Absorption spectra of DBSO (1.0  10-4 M) alone (black line) and recorded in the presence
of various Hg2+ concentrations (7.81  10-8  3.34  10-4 M) in 0.010 M SDS solution. Inset:
change in absorbance at 250 nm (A250) as a function of [Hg2+].
The interaction of the ligand with Hg2+ ions should be mainly located near the sulfur and nitrogen
atoms of the chelating region, being these atoms softer than oxygen and therefore more incline to
interact with the soft Hg2+ ions [34,35]. At Hg2+concentration close to that of DBSO (∼1  10-4 M),
no further absorbance increase at  > 240 nm was observed, in line with the complete complexation
of the ligand. The absorption continued to enhance only at wavelengths below 240 nm, assigned to
the mercury aquo complex by comparison with the absorption spectra recorded, in the absence of
receptor, upon addition of Hg2+ (1.67  10-7  3.36  10-4 M) in aqueous SDS solution (Fig. S1 in
the Supplementary Information). By plotting the absorbance at 250 nm (wavelength where only
DBSO-Hg2+ complex absorbed), as a function of [Hg2+] (insets in Fig. 1), a break point was
highlighted at 1 equivalent of Hg2+, providing evidence for a 1:1 binding stoichiometry. The
complexation constant (K), determined according to the procedure previously described [6], was
found to be 2.7  105 M-1. As observed in Fig. S2, a satisfactory fitting curve by Eq. (1) (where A0 is
8

for absorbance without metal, Alim is the limit value when the ligand is fully complexed, [Hg2+] and
[DBSO] refer to the analytical concentrations of the metal and ligand, respectively) was found, with
correlation coefficient of 0.9990, endorsing the 1:1 stoichiometry of the complex.
(1)
Taking into account that the sensorial system was examined with DBSO both at 1.0  10-4 and
3.0  10-4 M (see below), the changes in the absorption spectra of a more concentrated receptor
solution (3.0  10-4 M) mixed with Hg2+ (1.35  10-7  9.89  10-5 M) in SDS solution were also
recorded (Fig. S3). In this case, a plateau value was not achieved, since the spectra were recorded
up to 0.3 equivalents of Hg2+; anyway, a good fitting of absorbance at 250 nm vs. [Hg2+] data by eq
(1) was observed (Fig. S4), confirming the formation of the DBSO-Hg2+ complex with K value of
2.3  105 M-1 (correlation coefficient = 0.9976), value close to that calculated at DBSO lower
concentration.
3.2 Cation-induced changes in photophysical properties of the MA+/DBSO system in SDS micelles
As is evident in Fig. 2, the fluorescence emission intensity (max = 495 nm) of a SDS solution
of MA+ (5.3  10-6 M, kept constant in all experiments) was first quenched of 48% by addition of
1.0  10-4 M DBSO (black and green line, respectively) and then was turned back with gradual
addition of Hg2+ ions, without an appreciable shift in the emission maximum. At the excitation
wavelength used only the fluorophore absorbed (see absorption spectra of MA+ and DBSO in Fig.
S5). A plateau level was reached after titration by  1 equivalent of mercury ions with respect to the
4
receptor moles (see inset in Fig. 2). The fact that adding 1.0  10 M Hg2+ to the system in the
absence of DBSO resulted in a fluorescence quenching of just 1% (see Fig. S6) indicated that the
interaction between receptor and metal is indeed required for signal generation.
9

2.1x104
2.5x104
2.0x104
1.5x104
1.0x104
I495
1.8x104
1.5x104
1.2x104
-5
-4
-4
0.0
6.0x10
1.2x10
1.8x10
[Hg2+], M
5.0x103
0.0
400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 2. Fluorescence emission spectra of 5.3  10-6 M MA+ alone (black line), in the presence of 1.0
4
 10 M DBSO (green line) and various Hg2+ concentrations (7.81  10-8  1.73  10-4 M) in 0.010
M SDS solution; exc = 359 nm. Inset: change in intensity at 495 nm (I495) as a function of [Hg2+].
Thanks to the modular nature of the self-assembling sensor system in micellar medium, it was
possible to point out the effect of the receptor concentration on the fluorescence behavior of the
system, and so to tune the detection range just by the modification of the component ratio. Indeed,
for sensorial system MA+/DBSO, spectrofluorimetric titrations carried out at higher concentrations
of receptor with Hg2+ ions exhibited a similar spectral behavior, as observed in Fig. S7 and Fig. S8
and corresponding insets, where the plots of the fluorescence emission at concentration of 1.5  10-4
and 5.0  10-4 M receptor, respectively, are shown. For the sake of comparison, the I495 versus
[Hg2+] profiles at all the investigated concentrations of DBSO are collected in Fig. 3. An evidence
is that the quenching fluorescence percentage of MA+, calculated with respect to the fluorescence of
MA+ alone (indicated by open circle symbols in Fig. 3), dropped from 48 % in the presence of 1.0 
10-4 M DBSO to 67 and 91 % for 1.5  10-4 and 5.0  10-4 M DBSO solution, respectively. This
suggests that the electron transfer from DBSO to MA+ excited state [36], reaction assumed
10

plausible on the basis of their redox potentials (Eox = 0.94 V vs. SCE in CH3CN approximate equal
to that of triethylamine [37]; E*red = 2.32 V vs. SCE in CH3CN [18]) becomes more and more
efficient. This result is expected considering that the fluorophore, being charged, may be located in
the micelle-water interface (about 1 every 6 micelles, being 3  10-5 M the average micellar
concentration at the surfactant analytical concentration of 0.010 M [6]); accordingly, it will be
surrounded by an increasing number of ligands, equal to 3, 5, 17, with 1.0  10-4, 1.5  10-4 and 5.0
 10-4 M DBSO, respectively, suitable to turn off the acridinium fluorescence. The fluorescent
enhancement observed at all the DBSO concentrations in the presence of Hg2+ plausibly arise from
the metal coordination to the receptor chelating region, which removes in particular the amino
nitrogen of DBSO as the source responsible for the intermolecular quenching. Reasonably, the
Hg2+-amine interaction drops the lone pair energy, increasing oxidation potential, accordingly the
PET is less efficient. The emission intensity of MA+ turned on upon addition of Hg2+, reaching a
maximum value at  1 equivalent of metal, both at 1.5  10-4 and 5.0  10-4 M of DBSO (Fig. S7
and Fig. S8, respectively) as already observed at lower receptor concentration.
[DBSO], M
2.0x104
1.0x10-4
1.5x10-4
5.0x10-4
I495
1.5x104
1.0x104
5.0x103
0.0
2.0x10-4
4.0x10-4
6.0x10-4
8.0x10-4
0.0
[Hg2+], M
11

Fig. 3. Change in intensity at 495 nm (I495) as a function of Hg2+ concentration of MA+ (5.3  10-6
4
4
4
M) in the presence of 1.0  10 M (), 1.5  10 M () and 5.0  10 M () DBSO in 0.010 M
SDS solution; symbols , ,  represent I495 of MA+ alone.
Another important aspect of the profiles depicted in Fig.3 is the fluorescence enhancement
below 100% in all the examined cases; this means that the PET was not sufficiently suppressed by
mercuric ion binding. The restored fluorescence percentage, calculated with respect to the
fluorescence of MA+ alone, decreased by increasing DBSO concentration (with values of 88, 80
and 55 %), in line with the increase in efficiency of the PET process (see above). An explanation of
such behavior could be that four of the five ligating centers (two sulfurs and two oxygens) do not
contribute directly to remove the PET, rather they compete negatively by dispersing a fraction of
the ionic charge [38].
A very interesting feature of our system was that to show a significant increase in fluorescence
intensity (I495) already upon addition of Hg2+ in the micro and submicromolar range of
4
concentration (Fig. 3). Specially, in the presence of 1.0  10 M DBSO, plot I495 as a function of
Hg2+ concentration was clearly nonlinear at metal concentration lower than 1 equivalent, contrary to
what was observed by UV/vis titrations (Fig. 1). These experiments suggest that a ternary complex
DBSO-Hg2+-DBSO is selectively formed upon addition of lower concentrations of Hg2+, which
only a fluorescence signaling technique is able to detect because of its high sensitivity. The ability
of sulfide derivative ligands to form 2:1 (ligand:metal) complexes with mercuric ions is however
well known [39–41]. Further addition of metal (higher than 2 M) favored the formation of
complex DBSO-Hg2+ (1:1 binding stoichiometry), already rationalized by UV/vis investigation, that
led to a worse sensitivity of the sensor, as emphasized by the data shown in Fig. 3. The reason of
the more efficient ability of the 2:1 complex with respect to the 1:1 complex to remove the PET
process was researched by analyzing the geometry of the two structures. Thus, for a meaningful
discussion of the experimental results, DFT calculations were performed in gas phase for molecular
simulation of the geometrical structure of the complexes that the aza-thioether (with methyl instead
12

of dodecyl tail to simplify the calculation, named DBSO’) forms with Hg2+ ions. Fig. 4 depicts
DFT-optimized structures for the most stable conformers of complexes (DBSO’-Hg)2+ and
’
(DBSO2 -Hg)2+. Bond distances of interest are given in Table 1. The 1:1 complex was predicted to
present an almost linear coordination around the metal ion, with a Hg–S distances of 2.52 and 2.48
Å. Conversely, the Hg2+ complexed with two receptor molecules showed a distorted tetrahedral
coordination, with four S atoms from the DBSO’ ligands bonded to the Hg atom, with similar Hg–S
bond distances for a ligand (2.82 and 2.83 Å) and quite different for the other one (2.61 and 2.79
Å). Therefore, the Hg2+ complexes tend to be distorted by the formation of short bonds to more
covalently bound donor atoms, such as sulfur, and longer bonds to more ionically bound donor
atoms, as nitrogen and oxygen (see Table 1). The preferential bonding of Hg2+ ion to soft sulfur-
based ligands is in line with the HSAB (hard and soft acids and bases) classification [42]. The trend
between Hg(II)–S bond distance and Hg(II) coordination number was the same as observed for the
series of mononuclear thiolate complexes, Hg(SR)n [43]. Interestingly, the geometric distortion of
the second ligand should lead to the intramolecular hydrogen bonding interaction (depicted by an
oval frame in Fig 4b) between the hydroxyl and the nitrogen atom of DBSO’ molecule, which is
’
peculiar only of complex (DBSO2 -Hg)2+. The hydrogen bond length of 1.75 Å, quite shorter than
the averaged hydrogen bond distance in water (1.88 Å, in standard condition of pressure and
temperature [44]) highlights a significant bond strength. We hypothesize that just this hydrogen
interaction in the 2:1 complex, able to remove the PET from the donor nitrogen atom to excited
MA+, is responsible for the greater sensitivity of the sensor.
13

(a)
(b)
’
Fig. 4. Optimized structures of complexes (DBSO’-Hg)2+ (a) and (DBSO2 -Hg)2+ (b) (C = grey; O
= red; H = white; S = yellow).
Table 1
’
Optimized Hg-X (with X=S, N, O) bond distances for complexes (DBSO’-Hg)2+ .and (DBSO2 -
Hg)2+.a
Complex
d(Hg-S)
2.52, 2.48
d(Hg-N)
2.57
d(Hg-O)
2.50, 3.13
(DBSO’-Hg)2+
(DBSO’2-Hg)2+
2.61, 2.79, 2.82, 2.83
2.98, 4.01
3.72, 4.03, 5.58, 2.89
aValues, in Å, obtained at BLYP/TZ2P level using the scalar ZORA Hamiltonian.
Another intriguing evidence was that in the micromolar concentration range, plot I495 versus
[Hg2+] became more linear by increasing DBSO concentration, in parallel to a decrease of
sensitivity (Fig. 3). Indeed the slopes of the response curves, revealed that the signal change
4
induced by the same amount of Hg2+ (4 M) was ca. 10 and 50 times stronger at 1.0  10
M
4
4
DBSO compared to 1.5  10 and 5.0  10 M DBSO, respectively. Therefore, the sensitivity
decreases with the dilution of Hg2+ with respect to the receptor molecules in the micellar
aggregates, as it increases the probability that ions Hg2+ bind to receptors associated with micelles
devoid of fluorophore [21,45].
14

Equation
y = a + b*x
0.99997
Adj. R-Square
I/I01.20 Book1_Taratur Intercept
1
Value
Standard Error
--
a@7
Book1_Taratur Slope
a@7
1.28764E6
33526.12304
1.15
1.10
1.05
1.00
0.0
3.0x10-8 6.0x10-8 9.0x10-8 1.2x10-7 1.5x10-7
[Hg2+], M
Fig. 5. Fluorescence calibration curve (5.3  10-6 M MA+, 1.0  10-4 M DBSO in 0.010 M SDS
solution,  = 495 nm) used for the sensor assay in the Hg2+determination by linear plotting method
(I0 e I are the intensities of MA+ in the presence of DBSO without and with Hg2+, respectively);
()18 M Milli Q water and () tap water.
4
In optimal sensitivity condition, i. e. at [DBSO] = 1.0  10 M, the system was able to
generate an excellent linear response curve reported as I/I0 ratio (where I and I0 refer to the intensity
with and without Hg2+, respectively) as a function of Hg2+ concentration in the nM concentration
range (Fig. 5). For this purpose, a set of known various standard solutions of Hg2+ (8−120 nM),
prepared as described in Experimental Section, were measured and the calibration plot was
established by I/I0 ratio determination at 495 nm. The detection limit, calculated by the 3s-1
relationship, where  = standard deviation of blank response and s = slope of the linear regression,
4
4
was found 22 nM (against 2.0 M and 16 M at 1.5  10 and 5.0  10 M DBSO, respectively),
somewhat lower with that observed for other Hg2+-sensing systems based on the same motif
[46,47]. A response to [Hg2+] in the nM range was measured in real water samples (tap water). For
this purpose, the tap water sample solutions were prepared and the response percentage was of 98-
100 % compared with those in pure water, proving the sensing system tolerance to the presence of
other components (Fig. 5).
15

As regards the selectivity of the sensing system, it was evaluated by a comparative study
among a series of 10 metal ions (such as K+, Mg2+, Ca2+, Mn2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, and
Al3+) at a constant concentration of 1.0  10-5 M (Fig. 6).
1.4
I/I0
1.3
1.2
1.1
1.0
Hg2+
Mg2+
Ca2+
2+ Ag+ Cd2+
Al3+
no metal
K+
Mn2+ Ni
2+ Cu2+
Zn
Fig. 6. Plot of I/I0 (where I and I0 refer to the intensity of the MA+ (5.3  10-6 M)/DBSO (1.0  10-4
M) system in the presence and absence of metal ion, respectively, at 495 nm) recorded in 0.010 M
SDS solution upon addition of Hg2+ (1.0  10-6 M) and various cations (1.0  10-5 M), full bars. Not
filled bars represent I/I0 of MA+ with DBSO in the presence of the indicated metal ions, followed
by addition of 1.0  10-6 M Hg2+.
Except Al3+, only for Hg2+ (1.0  10-5 M) a significant increase in I/I0 ratio was observed, indicative
of the inability of other metal ions to form a complex with DBSO. By competition experiments,
carried out under identical conditions, fluorescence intensity was restored in the presence of Hg2+
(1.0  10-6 M) with all the metal ions examined. However, Al3+ was a significant interferent for the
mercury detection. We also performed a spectrophotometric titration (Fig. S9) with Al3+, in the
absence of MA+, and we compared the results with that obtained with Hg2+. Correlation of the
spectrophotometric data (A280 versus [Al3+], see inset of Fig. S9) by Eq. (1) consisting with the 1:1
complex model, allowed us to valuated a conditional K value of 5.8  104 M-1. Thus, a stability
constant of complex DBSO-Al3+ only 5 times smaller than that obtained for DBSO-Hg2+, together
16

with an enhancement of the “metal sponge effect” due to a stronger electrostatic interaction between
the anionic micellar surface and the more densely charged Al3+, make the selectivity of this system
towards Hg2+ not so good over Al3+.
3.3 Cation-induced changes in photophysical properties of the CA/DBSO system in SDS micelles
An intriguing scenario was observed when Hg2+ titrations were performed in the presence of
fluorophore CA. The fluorescence experiments, performed exciting selectively 5.0  10-6 M CA at
365 nm in 0.010 M SDS (absorption spectrum in Fig. S10), showed a lowering of the emission
intensity (maximum at 448 nm) when the DBSO amount increased from 1.0  10-4 to 3.0  10-4 M
(Fig. S11), as already observed in similar experiments using MA+ as a fluorophore. In particular,
the intensity was reduced of 13 and 31% of its initial value at 1.0  10-4 and 1.5  10-4 M DBSO,
respectively, against 48 and 67% obtained with MA+ at the same receptor concentrations. The PET
suppression therefore seems less effective with CA, most likely due to its lower reduction potential
in the singlet excited state (E*red = 1.46 V vs. SCE in CH3CN [19]). In the presence of 3.0  10-4 M
DBSO, the initial fluorescence enhancement of CA accompanying the mercury complexation of
DBSO plausibly removes the amine group from the electron transfer with the excited fluorophore
(Fig. S12). Curiously, for Hg2+ concentrations higher than ca. 6  10-5 M, a gradual decrease of the
fluorescence was instead observed up to 1.15  10-4 M (Fig. S13), never observed with MA+ as a
fluorophore. The lack of a significant effect of mercuric ions (from 1.00  10-7 to 2.00  10-4 M, see
Fig. S14) on the CA fluorescence emission in the absence of DBSO points out that the receptor
metal complex formation plays a decisive role. As already observed with MA+, the plot of I/I0 at
448 nm displaced as a function of [Hg2+] was clearly nonlinear (Fig. 7), exhibiting a much stronger
response at [Hg2+]/[DBSO] < 0.001 which was attributed to the formation of the ternary complex
DBSO-Hg2+-DBSO, though not expected on the basis of the UV/Vis titration (Fig. S3). The
subsequent rapid change of slope upon addition of [Hg2+] > 3  10-7 M suggests possibly the
17

involvement of a 1:1 binding stoichiometry. However, as reported above (Fig. S3), the absorption
spectra of 3.0  10-4 M DBSO mixed with Hg2+ from 1.35  10-7 to 9.89  10-5 M in SDS solution
confirmed the formation of the DBSO-Hg2+ complex.
1.6
I/I0
1.4
1.20
1.15
1.10
1.2
1.05
1.00
6.0x10-7 1.2x10-6 1.8x10-6
0.0
1.0
3.0x10-5 6.0x10-5 9.0x10-5 1.2x10-4
0.0
[Hg2+], M
Fig. 7 Plots of I/I0 at 448 nm as a function of Hg2+ concentration of CA (5.0  10-6 M) in the
4
presence of 3.0  10 M DBSO in 0.010 M SDS solution (I and I0 refer to the intensity with and
without Hg2+, respectively). Inset: enlargement of the 0  2  10-6 M concentration range.
The visible fall in fluorescence at [Hg2+]/[DBSO] higher than 0.2 clearly suggests that the PET
process is not sufficiently suppressed by mercury binding anymore, likely due to a charge dispersal
mechanism [38]. Specifically, the pairing ability of chloride anions to Hg2+ coordinated with the
receptor, leading to the DBSO-HgCl2 complex formation, becomes significant at higher Cl
concentration in the micellar interface [48,49]. This effect should cause a weakening of the
interaction of Hg2+ with the DBSO amine group, thus allowing quenching by a PET mechanism. At
this point one might wonder why with CA the fluorescence intensities passed through a maximum
and fell off by increasing the Hg2+ concentration, while with MA+ reached a plateau level, although
the same receptor was used (compare Fig. 7 with Fig. 3). In our opinion, such different behavior
18

may be rationalized taking into account that the self-assembly inside micelles involves the
consequent selective compartmentalization of the fluorophore and the receptor, both free and bound
to metal. We suppose the complex DBSO-Hg2+, made more lipophilic when the Hg2+ ion is further
coordinated to two chloride anions, to be more included in the micelle lipophilic core, where also
CA, being a hydrophobic fluorophore, should reside. This close proximity allows the ON-OFF
fluorescence switching of the CA/DBSO system in the presence of Hg2+, by a suitable charge
perturbation, able to restore an efficient PET. On the contrary, the acridinium moiety is likely
localized in the polar micelle-water interface (Stern layer), where the sulfate headgroups of
surfactant interacts with the softer acridinium ions rather than with sodium ions. In this case the
surfactant aggregates does not ensure the spatial closeness between fluorophore and complex
DBSO-Hg2+coordinated by chloride: the lipohilized ligand offers no quenching mechanisms,
accordingly the fluorescence of MA+ is maintained in “ON” condition.
The narrow range of Hg2+ concentrations, where the system CA/DBSO was able to generate a
linear response curve, is responsible for its not excellent performance in terms of sensitivity.
Selectivity study of CA/DBSO system was performed by recording the fluorescence spectra after
the addition of each metal ions. Fig. S15 shows the dependence of I/I0 as a function of cation
concentration (1.00  10-7, 1.00  10-6 and 1.00  10-5 M) evaluated for all metals already tested
with MA+ and in addition Pb2+and Fe3+. As already observed using MA+ as a fluorophore, only for
Hg2+ a significant increase in intensity ratio was detected, along with a relatively minor change in
the emission by Al3+ addition. Thus, as expected, the selectivity mainly was due to the ligand
choice.
4. Conclusions
The fluorophore/probe system depicted here is designed for Hg2+ ions and represents a new
OFF-ON fluorescent chemosensor. SDS micelles allow the inter-component communication
without the need for covalent linkage. This modular approach has allowed the easy change of the
19

fluorophore, the use of pure water instead of prevalently organic solvent mixtures, in addition to a
high solubility of the components. A tuning of the sensor sensitivity can be easily obtained by
changing the ligand to fluorophore ratio, without altering the recognition or reporter unit. Thus, the
receptor concentration acts as a regulatory element that determines the effectiveness at which the
presence of Hg2+ is transduced into signal. The main novelty of this work is that the interaction of
Hg2+ with DBSO allows to modulate the strength of the output signal simply by changing the
stoichiometry of the complexes between metal and receptor. The explanation of this experimental
evidence is supported by DFT computations. An additional feature is that coordination of Hg2+ to
covalently binding ligands, such as Cl, tends to weaken the interaction of mercury with the DBSO
N-donor, thus allowing quenching by PET. This occurs specially in the presence of a more
hydrophobic florophore, such as CA, and at higher concentrations of mercuric chloride. Therefore,
the signal transduction gives rise to response curves that are different from those typically observed
in both self-assembled and bonded sensorial systems. Despite the complexity of our system,
generated by assembly of three components, its performance in tap water has shown good
reproducibility compared to that performed in deionized water. This proves that the application of
this type of systems is a practicable way.
Acknowledgments
Thanks are due to the University of Perugia (Fondo Ricerca di Base 2018) for financial support.
20

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